| Liver cell damage and accumulation can cause liver disease,and the real-time detection and evaluation of liver cell damage and liver function is of great significance for the prevention,early diagnosis and treatment of liver disease.Glycocholic acid is one of the main components of bile acid.It is formed by the combination of cholesterol and glycine in the liver.Studies have shown that the excretion of glycocholic acid through the kidney is obvious,which is related to the severity of liver and gallbladder diseases.Therefore,the detection of glycocholic acid in urine has certain guiding value for clinical detection,and the clinical detection of glycocholic acid is mostly for taking venous blood.For pediatric patients and patients who are not convenient to take venous blood,the detection of glycocholic acid in their urine has its convenience and significance for disease diagnosis.In this study,monoclonal antibodies to GCA were first obtained by cell fusion.At the same time,surface plasmon resonance biosensors,immunochromatographic test strips,and chemiluminescence immunoassay methods based on monoclonal antibodies to GCA were established.The specific experimental content includes the following aspects:1.Preparation and characterization of monoclonal antibodyIn this experiment,the immune antigen GCA-BSA synthesized by the active lipid method was used to immunize female Balb/C mice.After cell fusion,a hybridoma cell line GCA-BSA-5H2 capable of stably secreting monoclonal antibodies was obtained.A large number of monoclonal antibodies were obtained by ascites induction method,and were firstly purified by caprylic acid-ammonium sulfate method,and then purified by Protein G column and monoclonal antibody subtyping kit to identify its subtype as IgG1 type.2.Establishing surface plasmon resonance biosensor based on glycocholic acid monoclonal antibodyIn this experiment,after the pre-immobilization,0.05 mg/mL GCA-OVA dissolved in sodium acetate buffer(pH=4.0)was injected into the chip at a flow rate of 10 ?l/min for 10 min.The established standard inhibition curve of the surface plasmon resonance biosensor of glycocholic acid,IC50 is 39.8ng/mL,linear range is 13.3-119.4ng/mL,R2=0.9993,LOD=2.5 ng/mL.Except for TCA,which has a cross-reaction rate of 40%,the cross-reaction rates of other structural analogues of glycocholic acid and corresponding antibodies are less than 10%.The surface plasmon resonance biosensor based on the established monoclonal antibody was veritied by recovery analysis.Three experimental urine samples were randomLy taken,and glyceric acid was added thereto to a concentration of 5 and 100 ?g/mL.The recovery rate was 79.7-119.7%.In addition,comparing the surface plasmon resonance biosensor with ELISA,the two have a strong correlation(R2=0.99).3.Establishing an immunochromatographic test strip based on the monoclonal antibodyIn this experiment,polyaniline-gold nanoparticles with an average particle size of about 115.45 ± 10.53 nm were prepared,and polyaniline-gold nanoparticles monoclonal antibody complexes were prepared using polyaniline-gold nanoparticles as a marker.1.5 mg/mL GCA-OVA was selected to determine the T-line coating concentration and 1:20000 dilution of the secondary antibody IgG was selected for the C-line coating concentration to establish an immunochromatographic test strip based on the glycine monoclonal antibody.The methodological investigation of the test paper on whether the content of GCA in 32 urine samples exceeded the standard,the results showed that the liver disease samples were all positive and the results of quantitative detection(ELISA)remained consistent.The test strip has good specificity,high reproducibility,and stable performance,and can be applied to the qualitative detection of glycocholic acid in urine.4.Chemiluminescence immunoassay method based on glycocholi acid monoclonal antibodyPolyaniline-gold nanoparticles(PPAuNP)were labeled with IgG-HRP,and the optimal working conditions of the ic-CLIA method were determined to be a coating time of 1.50 h,a blocking time of 0.75 h,and an antigen-antibody reaction time of 0.75 h.The reaction time between secondary antibody and antigen-antibody was 0.75 h,and the reaction time of luminescent substrate was 4min.Establish a standard curve for GCA.The detection range(IC15-IC85)of the established method was 161.99-61929.99 ng/mL,the IC50 value was 274.82,and the minimum detection limit(LOD,IC10)was 56.26 ng/mL.334 human urine samples were tested.The GCA concentrations in the urine samples of the chronic hepatitis,cirrhosis and liver tumor groups were 6.3 times,9.1 times,and 10.1 times(P<0.001),respectively,higher than the healthy group.The positive detection rates were 86.4%,78.9%,and 63.7%,respectively.In summary,this study prepared a hybridoma cell line GCA-BSA-5H2 that stably secreted monoclonal antibodies.Surface plasmon resonance biosensors,immunochromatographic analysis methods,and chemiluminescence immunoassay methods were established based on monoclonal antibodies to glycocholic acid.Through the qualitative examination of glycocholic acid in urine,it is possible to quickly determine whether the disease marker(glycolic acid)is abnormal in early screening,and the quantitative test results of glycocholic acid show that glycocholic acid is different in different liver diseases There is a significant difference in the content of uric acid,indicating that the level of glycocholic acid is closely related to the degree of liver pathological damage.Compared with clinical indicators AST,ALT,and AFP,glycocholic acid can better reflect potential chronic liver damage,and is superior to AST,ALT,and AFP indicators in judging cirrhosis and liver tumors.The immunological analysis methods studied in this experiment can effectively detect glycocholic acid and can monitor liver function. |
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